水稻稻种相关细菌及其生防种衣剂的研究
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摘要
了解种子上的微生物生态是获得良好生物防治效果的基础,开发与种子相关的拮抗菌来防治作物病害是生物防治的一种有效方法。本论文对南方水稻种子上的细菌进行了分离、鉴定,对其中具有致病性和拮抗性的细菌作了进一步研究；并以筛选的拮抗细菌为主要成分,开展了生防种衣剂的研制。对种衣剂的理化性状、贮藏性、作用机理及拮抗菌在水稻上的定殖作了研究。开发出有效防治水稻细菌性条斑病的生物种衣剂。
本试验对南方稻种上可培养细菌的群体性状开展了研究,共收集了南方稻区107个种子样品,分离获得428株细菌菌株。所有菌株分别进行了形态学、脂肪酸分析、致病性测定和体外拮抗试验,对其中具有致病性和拮抗性能的菌株作了16SrDNA分子生物学鉴定。在分离到的细菌中,革兰氏阴性菌占65.2%,革兰氏阳性菌占34.8%,共涉及20个属,40种菌。占优势的细菌属是Pseudomonas、Pantoea、Sphingomonas、Bacilllu、Microbacterium和Paenibacillus。致病性测定表明,大约占总分离数88%的细菌菌株和对照相比无明显症状；7%左右的菌株在接种的水稻上均能产生致病症状,并表现出较强的致病性,它们是Acidovorax avenae subsp. avenae、Burkholderia glumae、Pseudomonas fuscovaginae、Xanthomonas oryzae pv. oryzae和Xanthomonas oryzae pv. oryzicola;另外5%左右的菌株分别被鉴定为Bacilllu pumilus、Microbacterium barkeri、Pantoea agglomerans、Pantoea ananatis、Paenibacillus polymyxa、Pseudomonas aeruginosa、Pseudomonas syringae syringae和Xanthomonas sp.,在接种水稻后部分植株产生致病症状,但致病性表现相对较弱。体外拮抗试验表明,占总分离菌12.4%的细菌表现出对水稻细条病菌的拮抗性,涉及到14个种。筛选到的拮抗菌Pseudomonas putida A N51和Paennibacillus lentimorbus N72具有较强的拮抗水稻细菌性条斑病菌的能力,被用作生防制剂的开发。从稻种上分离到了泛菌属菠萝泛菌(Pantoa ananatis),分离频率达32.7%,是分离到的优势种,而此前国内尚未有该菌的报道,其中14.3%的菌株在接种水稻后产生了致病症状,P. ananatis在国外被认为是一种非传统的病原体,有待深入研究。
本试验以种子上筛选出来的拮抗菌缓病类芽孢杆菌N72和恶臭假单胞菌N51为有效成分,开展了生物种衣剂的研制,生物种衣剂N72表现出较好的生理生化性状、贮藏性和田间防治效果。在田间试验中,经N72生物种衣剂处理后,对细条病的防效达到72.71%,增产7.88%。在生防菌的定殖试验中发现用生物种衣剂N72和N51包衣种子,能够成功地将生防菌株N72和N51接种到水稻种子上,并随着种子的萌发、出苗和生长向上运转,定殖于水稻幼苗的根际、茎和叶部。播种60d后,在植株的各部位仍能检测到N72-Rif和N51-Rif,并且在根际的定殖量显著高于其它部位,说明良好的根际定殖能力也是拮抗菌株发挥生防作用的机制之一。在生物种衣剂的作用机理研究中发现用生防细菌N72和N51配置成的生防种衣剂包衣处理水稻种子后,水稻秧苗叶片内的POD酶活性和POO酶活性增加,而PAL酶活性没有变化。在接种了水稻细条病菌以后,三种酶的活性均出现了先下降再上升,达到高峰后又下降的趋势,生物种衣剂处理与对照相比,保持了较高的酶活性,说明在经过了生物种衣剂包衣种子处理后,植株获得了诱导抗性。本试验筛选的生防菌N72被鉴定为Paennibacillus lentimorbus,目前在国内还未见有该种细菌防治植物病原菌的报道。本试验研制的生物种衣剂已申请国家发明专利(专利申请号,200910154899.1)。
Knowledge of microbial ecology on plant seeds is the basis for effective biological control. Development of seed associated antagonistic bacteria to suppress plant diseases is one of the best ways in biocontrol. At present study the bacteria isolated from rice seeds of south China were identified and the pathogenic or antagonistic potential ones were further studied. A biological seed coating agent against bacterial blight and bacterial leaf streak was developed by the antagonistic bacteria screened from rice seeds as major component after its serial tests including physical, chemical and storage properties, mechanisms, colonization of the antagonistic bacteria in rice plants.
One hundred and seven rice seed samples were collected from south China and 428 bacterial strains were isolated. All the strains isolated were identified first by colony morphology, whole-cell fatty acid analysis, pathogenicity test on rice seedlings and antagonistic tests against major rice bacterial pathogens in vitro. The pathogenic and antagonic bacteria were further identified by 16SrDNA gene sequences. The bacteria isolated from rice seeds roughly consisted of 65.2%gram-negative and 38.2% gram-positive bacteria. It involved in 40 species of 20 bacterial genera. Predominant bacterial genera were identified as Pseudomonas, Pantoea, Sphingomonas, Microbacterium, Bacilllus and Paenibacillus. The pathogenicity tests showed that about 88% of total bacterial strains isolated had no any lesion after inoculation on rice seedlings. About 7% of the total strains induced remarkable symptoms on the inoculated rice seedlings, and were considered as true pathogens, including Acidovorax avenae subsp. avenae, Burkholderia glumae, Pseudomonas fuscovaginae, Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Another 5%of the strains were identified as Bacilllu pumilus, Microbacterium barkeri, Pantoea ananatis, Pantoea agglomerans, Paenibacillus polymyxa, Pseudomonas aeruginosa, Pseudomonas syringae syringae, Xanthomonas sp., respectively, which produced small lesions on rice seedings. About 12.4%of total strains showed in vitro antagonism against Xanthomonas oryzae pv. oryzicola, which involved in 14 species. Pseudomonas putida A N51 and Paennibacillus lentimorbus N72 were the 2 best strains showing good performance with larger inhibition zones, which were developed for biological control agents. In this study, Pantoa ananatis were first isolated from rice seeds in China with 32.7%isolation frequency as one of the dominant species. About 14.3%of P. ananatis induced disease symptoms on rice seedlings. P. ananatis is regarded as an unconventional plant pathogen occurring on previously unrecorded hosts in different parts of the world. It needs to be further studied.
Paennibacillus lentimorbus N72 and Pseudomonas putida A N51 mentioned above were used as major components for the biological seed coating agents in which their physical and chemical properties were examined in the experiment. Its pH, viscosity, sieve analysis, suspension rate, coating uniformity, and other indicators were in line with national quality standards of suspended seed coating product. Biological seed coating agent N72 showed excellent physical and chemical properties and storability. In the field experiment it significantly enhanced plant growth, and exhibited 72.71%control effectiveness against bacterial leaf streak of rice. The colonization experiment showed that both N72 and N51 could be successfully inoculated onto rice seeds, and colonized in the rhizosphere, roots, stems, and leaves of rice during the seed germination, emergence and plant growth. N72-Rif and N51-Rif could be found in the plant 60 days after sowing, and population of both strains were more stable and survived longer in rice rhizosphere than those in stem and leaves, indicating a good ability in rhizosphere colonization is one of the mechanisms of the antagonism strains in biocontrol. It was found in the mechanism research of biological seed coating agent that the activities of major defensive enzyme POD and POO were increased in rice leaves, and PAL did not changed after rice seed coated with biological agent N72 and N51. After inoculation of X. oryzae pv. oryzicola activities of the 3 enzymes were first decreased and then increased, reaching a peak then declining. The enzyme activities by biological seed coating treatment were higher than those of the control, indicating that induced disease resistance was acquired by rice plant after seed coating treatment. The biocontrol agent N72 used as major componet of biological seed coating agent were identified as P. lentimorbus which was no any report in China. The biological seed coating agent N72 have been applied for China innovation patent (Patent Name:A Paennibacillus lentimorbus strain and its seed coating agent; Patent Application Number: 200910154899.1).
本试验对南方稻种上可培养细菌的群体性状开展了研究,共收集了南方稻区107个种子样品,分离获得428株细菌菌株。所有菌株分别进行了形态学、脂肪酸分析、致病性测定和体外拮抗试验,对其中具有致病性和拮抗性能的菌株作了16SrDNA分子生物学鉴定。在分离到的细菌中,革兰氏阴性菌占65.2%,革兰氏阳性菌占34.8%,共涉及20个属,40种菌。占优势的细菌属是Pseudomonas、Pantoea、Sphingomonas、Bacilllu、Microbacterium和Paenibacillus。致病性测定表明,大约占总分离数88%的细菌菌株和对照相比无明显症状；7%左右的菌株在接种的水稻上均能产生致病症状,并表现出较强的致病性,它们是Acidovorax avenae subsp. avenae、Burkholderia glumae、Pseudomonas fuscovaginae、Xanthomonas oryzae pv. oryzae和Xanthomonas oryzae pv. oryzicola;另外5%左右的菌株分别被鉴定为Bacilllu pumilus、Microbacterium barkeri、Pantoea agglomerans、Pantoea ananatis、Paenibacillus polymyxa、Pseudomonas aeruginosa、Pseudomonas syringae syringae和Xanthomonas sp.,在接种水稻后部分植株产生致病症状,但致病性表现相对较弱。体外拮抗试验表明,占总分离菌12.4%的细菌表现出对水稻细条病菌的拮抗性,涉及到14个种。筛选到的拮抗菌Pseudomonas putida A N51和Paennibacillus lentimorbus N72具有较强的拮抗水稻细菌性条斑病菌的能力,被用作生防制剂的开发。从稻种上分离到了泛菌属菠萝泛菌(Pantoa ananatis),分离频率达32.7%,是分离到的优势种,而此前国内尚未有该菌的报道,其中14.3%的菌株在接种水稻后产生了致病症状,P. ananatis在国外被认为是一种非传统的病原体,有待深入研究。
本试验以种子上筛选出来的拮抗菌缓病类芽孢杆菌N72和恶臭假单胞菌N51为有效成分,开展了生物种衣剂的研制,生物种衣剂N72表现出较好的生理生化性状、贮藏性和田间防治效果。在田间试验中,经N72生物种衣剂处理后,对细条病的防效达到72.71%,增产7.88%。在生防菌的定殖试验中发现用生物种衣剂N72和N51包衣种子,能够成功地将生防菌株N72和N51接种到水稻种子上,并随着种子的萌发、出苗和生长向上运转,定殖于水稻幼苗的根际、茎和叶部。播种60d后,在植株的各部位仍能检测到N72-Rif和N51-Rif,并且在根际的定殖量显著高于其它部位,说明良好的根际定殖能力也是拮抗菌株发挥生防作用的机制之一。在生物种衣剂的作用机理研究中发现用生防细菌N72和N51配置成的生防种衣剂包衣处理水稻种子后,水稻秧苗叶片内的POD酶活性和POO酶活性增加,而PAL酶活性没有变化。在接种了水稻细条病菌以后,三种酶的活性均出现了先下降再上升,达到高峰后又下降的趋势,生物种衣剂处理与对照相比,保持了较高的酶活性,说明在经过了生物种衣剂包衣种子处理后,植株获得了诱导抗性。本试验筛选的生防菌N72被鉴定为Paennibacillus lentimorbus,目前在国内还未见有该种细菌防治植物病原菌的报道。本试验研制的生物种衣剂已申请国家发明专利(专利申请号,200910154899.1)。
Knowledge of microbial ecology on plant seeds is the basis for effective biological control. Development of seed associated antagonistic bacteria to suppress plant diseases is one of the best ways in biocontrol. At present study the bacteria isolated from rice seeds of south China were identified and the pathogenic or antagonistic potential ones were further studied. A biological seed coating agent against bacterial blight and bacterial leaf streak was developed by the antagonistic bacteria screened from rice seeds as major component after its serial tests including physical, chemical and storage properties, mechanisms, colonization of the antagonistic bacteria in rice plants.
One hundred and seven rice seed samples were collected from south China and 428 bacterial strains were isolated. All the strains isolated were identified first by colony morphology, whole-cell fatty acid analysis, pathogenicity test on rice seedlings and antagonistic tests against major rice bacterial pathogens in vitro. The pathogenic and antagonic bacteria were further identified by 16SrDNA gene sequences. The bacteria isolated from rice seeds roughly consisted of 65.2%gram-negative and 38.2% gram-positive bacteria. It involved in 40 species of 20 bacterial genera. Predominant bacterial genera were identified as Pseudomonas, Pantoea, Sphingomonas, Microbacterium, Bacilllus and Paenibacillus. The pathogenicity tests showed that about 88% of total bacterial strains isolated had no any lesion after inoculation on rice seedlings. About 7% of the total strains induced remarkable symptoms on the inoculated rice seedlings, and were considered as true pathogens, including Acidovorax avenae subsp. avenae, Burkholderia glumae, Pseudomonas fuscovaginae, Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Another 5%of the strains were identified as Bacilllu pumilus, Microbacterium barkeri, Pantoea ananatis, Pantoea agglomerans, Paenibacillus polymyxa, Pseudomonas aeruginosa, Pseudomonas syringae syringae, Xanthomonas sp., respectively, which produced small lesions on rice seedings. About 12.4%of total strains showed in vitro antagonism against Xanthomonas oryzae pv. oryzicola, which involved in 14 species. Pseudomonas putida A N51 and Paennibacillus lentimorbus N72 were the 2 best strains showing good performance with larger inhibition zones, which were developed for biological control agents. In this study, Pantoa ananatis were first isolated from rice seeds in China with 32.7%isolation frequency as one of the dominant species. About 14.3%of P. ananatis induced disease symptoms on rice seedlings. P. ananatis is regarded as an unconventional plant pathogen occurring on previously unrecorded hosts in different parts of the world. It needs to be further studied.
Paennibacillus lentimorbus N72 and Pseudomonas putida A N51 mentioned above were used as major components for the biological seed coating agents in which their physical and chemical properties were examined in the experiment. Its pH, viscosity, sieve analysis, suspension rate, coating uniformity, and other indicators were in line with national quality standards of suspended seed coating product. Biological seed coating agent N72 showed excellent physical and chemical properties and storability. In the field experiment it significantly enhanced plant growth, and exhibited 72.71%control effectiveness against bacterial leaf streak of rice. The colonization experiment showed that both N72 and N51 could be successfully inoculated onto rice seeds, and colonized in the rhizosphere, roots, stems, and leaves of rice during the seed germination, emergence and plant growth. N72-Rif and N51-Rif could be found in the plant 60 days after sowing, and population of both strains were more stable and survived longer in rice rhizosphere than those in stem and leaves, indicating a good ability in rhizosphere colonization is one of the mechanisms of the antagonism strains in biocontrol. It was found in the mechanism research of biological seed coating agent that the activities of major defensive enzyme POD and POO were increased in rice leaves, and PAL did not changed after rice seed coated with biological agent N72 and N51. After inoculation of X. oryzae pv. oryzicola activities of the 3 enzymes were first decreased and then increased, reaching a peak then declining. The enzyme activities by biological seed coating treatment were higher than those of the control, indicating that induced disease resistance was acquired by rice plant after seed coating treatment. The biocontrol agent N72 used as major componet of biological seed coating agent were identified as P. lentimorbus which was no any report in China. The biological seed coating agent N72 have been applied for China innovation patent (Patent Name:A Paennibacillus lentimorbus strain and its seed coating agent; Patent Application Number: 200910154899.1).
引文
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